Systems and methods for detecting a substance in bodily fluid

ABSTRACT

Various devices, systems and methods for determining a parameter of and/or detecting chemical and biological substances in bodily fluid are described herein. A device or system may include a substrate. An active sensor having an electrical characteristic and/or a control sensor may be disposed on the substrate. In certain variations, a differential between a first signal from the active sensor, and a second signal from the control sensor may be used to determine a parameter of the chemical or biological substance in the sample of bodily fluid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/599,190, filed on Jan. 16, 2015, which is a continuation of U.S.patent application Ser. No. 14/586,802, filed on Dec. 30, 2014, thecontents of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present systems and methods relate generally to devices, systems andmethods for detecting various parameters of a chemical or biologicalsubstance in bodily fluid.

BACKGROUND

Integration of biosensors on a small scale for e.g., in-home testing isincreasingly being favored by healthcare providers, however it has beena challenge for years. Optical-based biosensors require bulky detectionequipment and access to power supplies. Vibration sensitive biosensors(AFM, crystal-quartz balance etc.) cannot be built into a hand-held orportable device since background vibration will interfere with thesignal. Biosensors made from electrical components have been consideredas a good solution, however existing biosensors face several problemsincluding limitations caused by electrostatic screening in complexmedia. Several methods to circumvent this problem including sampledilution and pulsed electrical properties have been explored, resultingin additional sample processing steps and the dilution of the analyte,or adding complexity to the device design. In addition, reference andcalibration processes prior to use of the biosensors complicates theiruse.

Existing biosensors used to detect substances in bodily fluid sufferfrom a number of other limitations as well. For example, existingbiosensors may be utilized for analyte detection; however, due to theinability to control various environmental factors surrounding thesample of bodily fluid and the biosensor, signals associated with thisdetection are often not accurate, not reproducible and do not provide areliable or stable readout. Examples of existing biosensors can be foundin Pedro Estrela et al., Label-Free Sub-picomolar Protein Detection withField-Effect Transistors, Anal. Chem. 2010, 82, 3531-3536 and Eric Salmet al., Electrical Detection of Nucleic Acid Amplification Using anOn-Chip Quasi-Reference Electrode and a PVC REFET; Anal. Chem. 2014, 86,6968-6975, each of which is herein incorporated by reference.

As a result of the above limitations and restrictions, there is a needfor an improved device, system and method for detecting chemical andbiological substances in bodily fluid that minimizes or eliminates suchlimitations and restrictions.

BRIEF SUMMARY

Various electrical devices, systems and methods for determining aparameter of and/or detecting a chemical and biological substances inbodily fluid are described herein.

In one example, such a device includes a substrate having an activesensor; wherein the active sensor comprises a first electrical componenthaving an electrical characteristic; a functionalized structurecomprising a plurality of binding receptors each having at least onefunctional group associated therewith such that the functional group oneach of the binding receptors permits securing each of the bindingreceptors to a first layer of the sensor in a uniform manner, whereinthe binding receptor is configured to interact with the substance suchthat interaction of the substance with the functionalized structureresults in a change in the electrical characteristic of the firstelectrical component; and wherein the first electrical component isconfigured to generate a first signal upon the application of anelectrical stimulus, the first signal being indicative of the changedelectrical characteristic of the first electrical component and wherethe changed electrical characteristic allows determining the parameterof the substance.

A variation of the device can include a configuration where a distancebetween a binding site of the functionalized structure and the activesensor is minimized by the lack of an adhesion layer.

In such devices, the electrical stimulus can include altering anelectrical parameter selected from the group comprising: frequency,current, voltage, resistance, impedance, capacitance, conductivity,induction, threshold voltage, transconductance, subthreshold swing,piezo-resistivity, magnetic field, and electrical noise.

The binding receptor of the devices described herein can include aprotein selected from the group consisting of an engineered protein andan engineered scaffold protein.

In an additional variation, devices under the present disclosure canfurther include a second electrical component having an electricalcharacteristic, wherein interaction of the substance with the activesensor or the control sensor does not result in a change in theelectrical characteristic of the second electrical component.

The improved binding receptors described herein can include a functionalgroup that is directly engineered onto the binding receptor.

Variations of the device include the functionalized structure beingpositioned on the substrate or active sensor or in a location separatefrom the substrate, wherein the functionalized structure is configuredto bind the substance, wherein binding of the substance with thefunctionalized structure produces or results in the release of one ormore ions which are detected by the first electrical component or causea change in the electrical characteristic of the first electricalcomponent.

In an additional variation, the functionalized structure can bepositioned on the substrate or active sensor or in a location separatefrom the substrate, wherein the functionalized structure is configuredto bind the substance, wherein binding of the substance with thefunctionalized structure changes the electrical characteristic of thefirst electrical component.

The substrate can be positioned in a first location and thefunctionalized structure can be positioned in a second location separatefrom the substrate, wherein the functionalized structure is configuredto bind to the substance, wherein after binding the substance undergoesa reaction which changes the electrical characteristic of the firstelectrical component of the active sensor.

The substances screened in the present devices can include atherapeutic, drug, biological moiety, chemical moiety, protein, toxin,ion, antibody, peptide, oligonucleotide, pathogen (e.g., bacteria,viruses, fungi), cells (tumor cells, blood cells, other bodily cells),or ligands.

The high-k dielectric layer described herein can include, but is notlimited to, aluminum oxide, titanium oxide, zirconium oxide, yttriumoxide, silicon oxide, tantalum oxide, hafnium oxide and silicon nitride.The immobilization structure can further include nanoparticles and/or ametal layer for adhering to the layer.

In addition, the devices described herein can comprise a disposablestructure, wherein a plurality of active and control sensors arepositioned on the disposable structure.

In an additional variation, devices for determining a parameter of asubstance in a test substance can include a substrate; a firstelectrical component having an electrical characteristic, the firstelectrical component comprising an active sensor having a coveringlayer; a functionalized structure comprising a binding receptor freefrom endogenous functional groups and having a targeted functionalgroup, where the targeted functional group immobilizes the bindingreceptor to the covering layer such that the binding receptor extends nomore than 5 nm from the covering layer to minimize a screening length ofthe functionalized structure, where the binding receptor is configuredto interact with the test substance such that interaction of the testsubstance with the functionalized structure alters the electricalcharacteristic of the first electrical component; and whereinapplication of an electrical stimulus to the first electrical componentgenerates a first signal from the first electrical component beingindicative of the changed electrical characteristic of the firstelectrical component. In additional variations of the device, thebinding receptor can extend beyond 5 nm as needed while still providingan acceptable screening length for the desired application.

The present disclosure also includes methods for determining a parameterof a substance in a test sample. In one variation, such a methodincludes providing a substrate having an active sensor covered in afirst layer and having a first electrical component, wherein the activesensor comprises at least one functionalized structure in electricalcommunication with the active sensor and wherein the control sensorcomprises a second electrical component having an electricalcharacteristic, where the functionalized structure includes a bindingreceptor having a functional group coupled thereto such that thefunctional group secures the binding receptor to the functionalizedstructure without the need of an adhesion layer to minimize a distancebetween a surface of the functionalized structure and the active sensor;binding the substance to the functionalized structure, wherein afterbinding, the functionalized structure affects the first electricalcomponent to produce a changed electrical characteristic, where thechanged electrical characteristic varies from the electricalcharacteristic; determining a comparison between the electricalcharacteristic and the changed electrical characteristic; using thecomparison to determine at least one parameter of substance in the testsample; and producing an output of the at least one parameter.

In an additional variation, the method can include a functionalizedstructure that is positioned on the substrate or active sensor or in alocation separate from the substrate, wherein the functionalizedstructure binds the substance, wherein binding of the substance with thefunctionalized structure produces or results in the release of one ormore ions which are detected by the first electrical component or causea change in the electrical characteristic of the first electricalcomponent.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates a variation of a system for detecting a chemical orbiological substance in bodily fluid including a substrate having activeand control sensors and an analyzer, and a reader.

FIG. 2 illustrates a schematic diagram of the signal detection and readout of the system according to FIG. 1.

FIG. 3A illustrates a substrate having an active sensor and a controlsensor where functionalized structures are disposed on the activesensor.

FIG. 3B illustrates a side view of the substrate of FIG. 3A, where atarget chemical or biological substance is bound to a functionalizedstructure and undergoes a reaction which produces ions which diffuse tothe surface of the active sensor.

FIG. 4A illustrates a substrate having an active sensor and a controlsensor where functionalized structures are disposed on the substrate,adjacent to the active sensor.

FIG. 4B illustrates a side view of the substrate of FIG. 4A, where atarget chemical or biological substance is bound to the functionalizedstructure and undergoes a reaction which produces ions which diffuse tothe surface of the active sensor.

FIG. 5 illustrates a side view of an active sensor having functionalizedstructures disposed thereon, where first and second moieties of achemical or biological substance undergo a competing reaction whichproduces ions which diffuse to the surface of the active sensor.

FIG. 6 illustrates various functionalization schemes and secondaryreactions that a bound chemical or biological substance may undergo.

FIG. 7 illustrates various reactions that a bound chemical or biologicalsubstance may undergo.

FIG. 8A provides an illustrative example of the differences between abinding site of conventional receptor molecule and binding receptorswith a minimized binding site length, which lies within the or in closeproximity to the screening length instead of outside.

FIG. 8B provides an example of an endogenous functionalized group on aconventional receptor molecule and a binding receptors with anengineered functionalized group that immobilizes the binding receptor tothe sensor.

FIG. 9 shows one example of a FET sensor used to determine a parameterof a substance in a test sample.

FIG. 10A illustrates an example of a device that increases thereliability of measurement of a substance in the test sample using asignal comparison between two sensors.

FIG. 10B illustrates additional variations of functionalized sensors anda reference sensor.

FIGS. 11A to 11C provide illustrations of various binding receptors andsecondary reactions that can be measured by the sensor.

DETAILED DESCRIPTION

Variations of the devices are best understood from the detaileddescription when read in conjunction with the accompanying drawings. Itis emphasized that, according to common practice, the various featuresof the drawings may not be to-scale. On the contrary, the dimensions ofthe various features may be arbitrarily expanded or reduced for clarityand not all features may be visible or labeled in every drawing. Thedrawings are taken for illustrative purposes only and are not intendedto define or limit the scope of the claims to that which is shown.

In certain variations, a device, e.g., an electrical device orbiosensor, for determining a parameter of and/or for detecting achemical or biological substance in bodily fluid is provided. The deviceincludes a substrate. One or more active sensors and one or more controlsensors may be disposed on the substrate.

The active sensor includes one or more first electrical componentshaving an electrical characteristic or property. One or morefunctionalized structures are disposed on, near or in a vicinity of thesubstrate or active sensor. The functionalized structure is configuredto interact with, e.g., couple with or bind, the chemical or biologicalsubstance, e.g., one or more moieties of the chemical or biologicalsubstance. The interaction of the chemical or biological substance withthe functionalized structure results in a change in the electricalcharacteristic or property of the electrical component of the activesensor.

The control sensor comprises one or more second electrical componentshaving an electrical characteristic. However, the control sensor isconfigured such that interaction of the chemical or biological substancewith the active sensor and/or the control sensor does not result in achange in the electrical characteristic of the second electricalcomponents of the control sensor.

The first electrical component of the active sensor produces a signal,where the signal is indicative of the change in the electricalcharacteristic of the first electrical component caused by theinteraction of the functionalized group with the chemical or biologicalsubstance in the bodily fluid. For example, the signal may be indicativeof a changed current, voltage, capacitance or other electricalcharacteristic. The interaction between the functionalized group and thechemical or biological substance may take place on the active sensor oroff the active sensor or substrate in a separate location.Simultaneously, the second electrical component of the control sensorproduces a control signal. The differential between the signal from thefirst electrical component of the active sensor, and the signal from thesecond electrical component of the control sensor is used to determinethe parameter of the chemical or biological substance in the sample ofbodily fluid. Indeed, the device, via the simultaneous use and detectionof a control sensor, provides a self-calibration.

The differential signal may be used to determine a variety of parametersor characteristics of the chemical or biological substance, or to detectthe presence of the chemical or biological substance. In certainvariations, the differential signal may be used to determine theconcentration of the chemical or active substance. The differentialsignal may be used to determine the concentration of a variety of ionspresent in the chemical or biological substance or in the sample ofbodily fluid. For example, the differential signal may be used todetermine the pH of the chemical or biological substance.

In certain variations, the differential between the signal produced bythe active sensor and the signal produced by the control sensor isindicative of, corresponds to, or is used to determine the concentrationof the chemical or biological substance in the sample of bodily fluid.Indeed, the control sensor signal may correspond to a knownconcentration, such that the differential between the control signal andthe signal from the active sensor may be used to deduce or determine theconcentration of the chemical or biological substance in the sample ofbodily fluid.

The comparison between the signal of the active sensor and the signal ofthe control sensor may be the differential change (ΔS) in an electricalcharacteristic, e.g., current, voltage, capacitance, resistance orthreshold voltage, of the active sensor (S1) vs the control sensor (S2),where the comparison yields a differential signal is (ΔS=S1−S2).

In other variations, the signal produced by the active sensor, which isindicative of the changed electrical characteristic or the change in theelectrical characteristic of the first electrical component which changeoccurs as a result of the interaction of the functionalized group withthe chemical or biological substance, may be indicative of a changedcurrent, voltage, capacitance or other electrical characteristic. Forexample, the signal or detected current may correspond to a knownconcentration, such that the concentration can be deduced from thedetected current, or current change, or the concentration may be deducedfrom a detected change in another electrical characteristic or propertywhich corresponds to a known concentration.

The functionalized structure is configured to interact with the chemicalor biological substance of the sample of bodily fluid. As describedsupra, the interaction of the chemical or biological substance with thefunctionalized structure may result in a change in the electricalcharacteristic or property of the electrical component of the activesensor.

In any of the variations described herein, the electrical components mayinclude a transistor, a capacitor, a resistor or an inverter or anyother suitable electrical component known to persons have ordinary skillin the art.

A variety of interactions between the chemical or biological substanceand the functionalized structure and/or related reactions arecontemplated, where such interactions and/or reactions result in achange, or cause a change, in the electrical characteristic or propertyof the electrical component of the active sensor.

In one variation, one or more functionalized structures are disposed onthe active sensor and are configured to bind to the chemical orbiological substance. The binding of the chemical or biologicalsubstance by the functionalized structure results in a change in anelectrical characteristic of the electrical component of the activesensor. For example, the binding of a charged moiety or ion of achemical or biological substance by a functionalized structure mayresult in an increase or decrease in charge density on or in a vicinityof the active sensor or a change in current.

In another variation, one or more functionalized structures are disposedon the substrate or the active sensor, and are configured to bind to thechemical or biological substance. The binding of the chemical orbiological substance by the functionalized structure produces one ormore ions which diffuse to the surface of the active sensor and cause achange in an electrical characteristic of the electrical component ofthe active sensor. For example, the produced ions may come into contactwith, bind or otherwise interact with the active sensor, e.g., causingan increase or decrease in charge density of the active sensor or achange in current. Optionally, the functionalized structures may bedisposed in another location, separate from the substrate. The producedions may then flow over the surface of the active sensor, and interactwith the active sensor, causing a change in the electricalcharacteristic of the electrical component of the active sensor.

In another variation, one or more functionalized structures are disposedon the substrate or the active sensor, and are configured to bind to thechemical or biological substance. The bound chemical or biologicalsubstance undergoes a reaction with one or more reagents, therebyproducing one or more ions which diffuse to the surface of the activesensor and cause a change in an electrical characteristic of theelectrical component of the active sensor. For example, the producedions may come into contact with, bind or otherwise interact with theactive sensor, e.g., causing an increase or decrease in charge densityof the active sensor or a change in current. Optionally, thefunctionalized structures may be disposed in another location, separatefrom the substrate. Where the reaction takes place in a location whichis separated from the substrate, the produced ions may flow to and overthe surface of the active sensor, and interact with the active sensor,causing a change in the electrical characteristic of the electricalcomponent of the active sensor.

In another variation, the one or more functionalized structures may bein the form of a permeable membrane or other filter, which is disposedon the active sensor. The membrane or filter is configured to allow forthe passage of the target chemical or biological substance or a producedion, such that the substance or ion may interact with the active sensor,while the membrane or filter blocks or restricts the passage of othermoieties or ions, e.g., based on size or other property. The ions may beproduced as a result of the binding of the chemical or biologicalsubstance by the functionalized structure or as a result of a reactionbetween a bound substance and a reagent. The ions may diffuse or flow,from a local or remote location, over the surface of the active sensor,after passing through a membrane of filter, and cause a change in anelectrical characteristic of the electrical component of the activesensor. For example, the produced ions may come into contact with, bindor otherwise interact with the active sensor, e.g., causing an increaseor decrease in charge density of the active sensor or a change incurrent. Optionally, the membrane, filter or other functionalizedstructure may capture the target substance or ions, but allow thepassage of other non-target ions. Optionally, a membrane, filter orother functionalized structure may block background charge, where acharge or lack of charge may be detected when a particle flows through amembrane and past the sensor.

In certain variations, a device, e.g., an electrical device or abiosensor, for determining a parameter of and/or for detecting achemical or biological substance in bodily fluid is provided. The deviceincludes a substrate. One or more active sensors may be disposed on thesubstrate.

The active sensor includes one or more first electrical componentshaving an electrical characteristic or property. One or morefunctionalized structures are disposed on, near or in a vicinity of thesubstrate or active sensor. The functionalized structure is configuredto interact with, e.g., couple with or bind, the chemical or biologicalsubstance, e.g., one or more moieties of the chemical or biologicalsubstance. The bound chemical or biological substance undergoes areaction thereby producing a product. The product interacts with thefirst electrical component which results in a change in the electricalcharacteristic of the first electrical component. A signal from thefirst electrical component, the signal being indicative of the changedelectrical characteristic or the change in the electricalcharacteristic, may be used to determine the parameter of the chemicalor biological substance in the sample of bodily fluid.

The signal from the first electrical component of the active sensor maybe used to determine a variety of parameters or characteristics of thechemical or biological substance, or to detect the presence of thechemical or biological substance. In certain variations, the signal maybe used to determine the concentration of the chemical or activesubstance. The signal may be used to determine the concentration of avariety of ions present in the chemical or biological substance. Forexample, the signal may be used to determine the pH of the chemical orbiological substance.

In certain variations, the signal produced by the active sensor isindicative of, corresponds to, or is used to determine the concentrationof the chemical or biological substance in the sample of bodily fluid.Indeed, the signal may correspond to a known concentration, such thatthe signal from the active sensor may be used to deduce theconcentration of the chemical or biological substance in the sample ofbodily fluid.

In other variations, the signal produced by the active sensor, which isindicative of the changed electrical characteristic or the change in theelectrical characteristic of the first electrical component, may beindicative of a changed current, voltage, capacitance or otherelectrical characteristic. For example, the signal or detected currentmay correspond to a known concentration, such that the concentration canbe deduced from the detected current.

In any of the variations described herein, the electrical components mayinclude a transistor, a capacitor, a resistor or an inverter or anyother suitable electrical component known to persons have ordinary skillin the art.

A variety of interactions between the chemical or biological substanceand the functionalized structure and/or related reactions arecontemplated, where such interactions and/or reactions result in achange, or cause a change, in the electrical characteristic or propertyof the electrical component of the active sensor.

In one variation, one or more functionalized structures are disposed onthe substrate or the active sensor, and are configured to bind to thechemical or biological substance. The binding of the chemical orbiological substance by the functionalized structure produces one ormore ions which diffuse to the surface of the active sensor and cause achange in an electrical characteristic of the electrical component ofthe active sensor. For example, the produced ions may come into contactwith, bind or otherwise interact with the active sensor, e.g., causingan increase or decrease in charge density of the active sensor or achange in current. Optionally, the functionalized structures may bedisposed in another location, separate from the substrate. The producedions may then flow over the surface of the active sensor, and interactwith the active sensor, causing a change in the electricalcharacteristic of the electrical component of the active sensor.

In another variation, one or more functionalized structures are disposedon the substrate or the active sensor, and are configured to bind to thechemical or biological substance. The bound chemical or biologicalsubstance undergoes a reaction with one or more reagents, therebyproducing one or more ions which diffuse to the surface of the activesensor and cause a change in an electrical characteristic of theelectrical component of the active sensor. For example, the producedions may come into contact with, bind or otherwise interact with theactive sensor, e.g., causing an increase or decrease in charge densityof the active sensor or a change in current. Optionally, thefunctionalized structures may be disposed in another location, separatefrom the substrate. Where the reaction takes place in a location whichis separated from the substrate, but the produced ions may flow over thesurface of the active sensor, and interact with the active sensor,causing a change in the electrical characteristic of the electricalcomponent of the active sensor.

In one variation, the bound chemical or biological substance (on thesubstrate or in a remote location) may undergo a reaction which producesor results in the release of one or more ions which flow over the activesensor and cause a change in pH or other ion concentration. For example,the change in pH may be detected by the first electrical component ofthe active sensor, which may have a proton sensitive layer disposedthereon. The change in pH or other ion concentration may cause a changein the electrical characteristic of the first electrical component.

In another variation, the one or more functionalized structures may bein the form of a permeable membrane or other filter, which is disposedon the active sensor. The membrane or filter is configured to allow forthe passage of the target chemical or biological substance or a producedion, such that the substance or ion may interact with the active sensor,while the membrane or filter blocks or restricts the passage of othermoieties or ions, e.g., based on size or other property. The ions may beproduced as a result of the binding of the chemical or biologicalsubstance by the functionalized structure or as a result of a reactionbetween a bound substance and a reagent. The ions may diffuse or flow,from a local or remote location, over the surface of the active sensor,after passing through a membrane of filter, and cause a change in anelectrical characteristic of the electrical component of the activesensor. For example, the produced ions may come into contact with, bindor otherwise interact with the active sensor, e.g., causing an increaseor decrease in charge density of the active sensor or a change incurrent. Optionally, the membrane, filter or other functionalizedstructure may capture the target substance or ions, but allow thepassage of other non-target ions. Optionally, a membrane, filter orother functionalized structure may block background charge, where acharge or lack of charge may be detected when a particle flows through amembrane and past the sensor.

The various reactions described herein may allow indirect detection,where the product of the reaction may diffuse to the surface of thesensor where it interacts with the sensor. This helps circumventelectrostatic screening issues that might otherwise arise.

In certain variations, the substrate may also include a control sensor(as described supra). The control sensor includes a second electricalcomponent having an electrical characteristic. Binding of the chemicalor biological substance to the active sensor or the control sensor doesnot result in a change in the electrical characteristic of the secondelectrical component of the control sensor.

The active and control sensor are used simultaneously, where both aredisposed on the substrate. A differential between a signal from thefirst electrical component of the active sensor, the signal beingindicative of the changed electrical characteristic or the change in theelectrical characteristic, and a signal from the second electricalcomponent of the control sensor may be used to determine the parameterof the chemical or biological substance in the sample of bodily fluid,as described supra.

In certain variations, the devices described herein may be part of asensor or detection system. For example, the device may include, becoupled to, or be in communication with an analyzer. The analyzer may beconfigured to analyze the signals received from the first and/or secondelectrical components of the device or biosensor and to determine thedifferential between the signals. The system may also include a reader,where the reader includes, is coupled to or is in communication with theanalyzer. The reader is configured to provide an electrical read-out ofthe analyzed signals and/or the determined parameter, based on thedifferential signal.

The analyzer may be used to receive or read the signal from the activeand control sensors and to perform smart operations to convertmeasurements to accurate signal readouts and results for both sensors.The analyzer may include an analog/digital converter and/or amultiplexer. The analyzer may be used to provide a comparison, such as acomparison of signals or other differential readout (See FIG. 2), and/orfor amplifying the signals. The analyzer may include one or moresource-meters or other electronics to apply a voltage or current, toapply a pulsed signal, to read-out a voltage, to read-out a current,and/or to read out a resistance and/or capacitance change or otherelectrical characteristic change.

The reader may connect to or be coupled to the device, to the analyzer,and/or to the device having an analyzer incorporated therein. The devicemay be in the form of a strip having a plurality of sensors as describedsupra. The reader may receive input from the analyzer, and may be usedto visualize the detected and analyzed signals or results from thesensors of the device in a simple and user friendly way. The reader mayinclude one or more source-meters or other electronics to apply avoltage or current, to apply a pulsed signal, to read-out a voltage, toread-out a current, and/or to read out a resistance and/or capacitancechange or other electrical characteristic change.

In one variation, a device having one or more sensors and an analyzer,e.g., a device strip having a plurality of sensors as described herein,may be inserted into the reader to provide a user-friendly read-outregarding a parameter, (e.g., concentration) of the chemical orbiological substance detected by the sensor. The reader may applydifferent voltages or currents or other electronic properties to thedevice strip or analyzer and it may receive or provide an output whichmay be visualized by a user in a simple and effective manner.Optionally, the reader may have capabilities or be configured tocommunicate via Bluetooth or Wi-Fi or via other wired or wirelessmechanisms or modes of communication to one or more other device.Optionally, a controller may be provided, where the controller iscoupled to or in communication with the device, e.g., the sensors,analyzer, and/or a reader, such that the controller may be used tocontrol or program the functionality of the device, including thesensors and/or the analyzer. The controller may be coupled to theanalyzer or be integrated in the reader. In certain variation, acontroller may be located in the substrate (chip), analyzer or thereader.

In certain variations, methods for determining a parameter of a chemicalor biological substance in a sample of bodily fluid include one or moreof the following steps. Providing a substrate having an active sensorand a control sensor, wherein the active sensor comprises a firstelectrical component having an electrical characteristic, wherein atleast one functionalized structure is disposed on the substrate or theactive sensor or in a location remote or separated from the substrateand active sensor, and wherein the control sensor comprises a secondelectrical component having an electrical characteristic. Coupling orbinding the chemical or biological substance to a functionalizedstructure, wherein the bound chemical or biological substance undergoesa reaction thereby producing a product. Interacting the product with thefirst electrical component which results in a change in the electricalcharacteristic of the first electrical component, while not resulting ina change in the electrical characteristic of the second electricalcomponent. Comparing a first signal and a second signal, where the firstsignal is from the first electrical component of the active sensor, thefirst signal being indicative of the changed electrical characteristicor a change in the electrical characteristic, and the second signalbeing from the second electrical component of the control sensor. Usingthe differential to determine the parameter of the chemical orbiological substance in the sample of bodily fluid. Providing anelectronic read-out of the determined parameter, based on thedifferential.

The functionalized structure may be positioned on the substrate oractive sensor or in a location separate from the substrate, wherein thefunctionalized structure couples or binds the chemical or biologicalsubstance. Binding or coupling of the chemical or biological substancewith the functionalized structure produces or results in the release ofone or more ions which are detected by the first electrical component orcause a change in the electrical characteristic of the first electricalcomponent.

The bound or coupled chemical or biological substance may undergo areaction with one or more reagents which produces or results in therelease of one or more ions which are detected by the first electricalcomponent or cause a change in the electrical characteristic of thefirst electrical component.

The bound or coupled chemical or biological substance may undergo areaction which produces or results in the release of one or more ionswhich cause a change in a pH or other ion concentration, wherein thechange in pH other ion concentration is detected by the first electricalcomponent or causes a change in the electrical characteristic of thefirst electrical component.

The bound chemical or biological substance may undergo a reaction in afirst location which produces or results in the release of one or moreproducts or ions which flow to the active sensor on the substratepositioned in a second, separate location, where the products or ionsare detected by the first electrical component of the active sensor orcause a change in the electrical characteristic of the first electricalcomponent of the active sensor.

The following documents are incorporated herein by reference in theirentirety: Hammock, M. L. et al. Electronic readout ELISA with organicfield-effect transistors as a prognostic test for preeclampsia.; U.S.Provisional Pat. App. No. 61/907,363; and Mathias, W. et al. SelectiveSodium Sensing with Gold-Coated Silicon Nanowire Field-EffectTransistors in a Differential Setup. ACS Nano 7, 5978-5983 (2013).

The devices, systems or methods for determining a parameter of and/orfor detecting a chemical or biological substance in bodily fluiddescribed herein may be utilized with various bodily fluids to detectvarious parameters of various substances.

Bodily fluid may include, e.g., blood, urine, saliva, tears, ejaculate,odor or other body fluids. Detected substances can include, e.g.,hormones, different pathogens, proteins, antibodies, various drugs ortherapeutics or other chemical or biological substances. Detected ordetermined parameters may include, e.g., pH changes, lactose changes,changing concentration, particles per unit time where a fluid flows overthe device for a period of time to detect particles, e.g., particlesthat are sparse, and other parameters.

The various devices, systems or methods described herein may include oneor more of the following features described below.

In certain variations, a plurality of conductors may be coupled to theactive sensor and/or a plurality of conductors may be coupled to thecontrol sensor. The conductors may be adapted to be electrically coupledto a reader for obtaining an electrical reading from the electricalcomponents of the active and control sensors.

The chemical or biological substance may include, but not be limited to,a variety of substances, e.g., any substance suitable for detection ormonitoring, such as, a therapeutic, drug, biological moiety, chemicalmoiety, protein, ion or antibody.

A variety of functionalized structures may be utilized, e.g. proteins,peptides, antibodies, or chemical moieties. Any of these functionalizedstructures may be configured or suitable to bind to a therapeutic, drug,biological moiety, chemical moiety, protein, antibody, or ion in thesample of bodily fluid. In other variations, types of functionalizedstructures include, but are not limited to, a permeable membrane,hydrogel or other filter, e.g., PVC.

In one variation, a functionalized structure may include a bindingreceptor immobilized on the surface of the active sensor, and thebinding receptor (e.g. antibody, protein, peptide) may be capable ofbinding to any of the chemical or biological substances describedherein.

In certain variations, an immobilization structure may be disposed on orin a vicinity of a substrate or active sensor of a device, and afunctionalized structure may be coupled to the immobilization structure.For example, the immobilization structure may include a high-Kdielectric layer such as an atomic layer deposition (“ALD”) or any othertechnique can be used to deposit the layer (e.g., such as Growing anoxide layer or nitride etc. on top of the sensor). The high-κ dielectriclayer may include, but not be limited to, aluminum oxide, titaniumoxide, zirconium oxide, yttrium oxide, silicon oxide, tantalum oxide,hafnium oxide and silicon nitride. Optionally, the immobilizationstructure may include at least a portion made up of nanoparticles and/ora metal layer for adhering to the layer.

In certain variations, the control sensor may be passivated. Forexample, the control sensor may include a passivation structure such asa self-assembled monolayer (SAM), metal, or polymer layer. The SAM mayinclude alkane or aromatic thiols, aromatic silanes, or any chemicalentity having a terminal group that is covalently attached to a surface,a spacer group having a hydrocarbon, or a head group, e.g., such as,—COOH, —CH3, —SH, —NH2, long chain alkyl of any length or aromatic and—OH. The SAM layer can include a silane where the silanes bearing along, hydrophobic chain of any length e.g. long alkyl chain of anylength, e.g. Octadecyldimethylmethoxysilane.

In any of the variations described herein, the device or substrate maybe in the form of a disposable structure. The disposable structure mayinclude a plurality of active and/or control or passivated sensorspositioned thereon. In certain variations, the device or strip mayinclude an analyzer and/or a reader incorporated therein.

The device or strip may include a plurality of active sensors and/orcontrol sensors. The signals generated by each sensor, e.g., signalsresulting from a change to an electrical characteristic of the activesensors, may be read out. The average of all the determined parametervalues based on each sensor, e.g., the concentration of a substance, maythen be calculated or deduced.

In any of the various devices, systems or methods described herein,various electrical components or sensors may be utilized. The electricalcomponent or sensor may be any suitable transistor, e.g. an OFET(organic field effect transistor) or FET (field effect transistor). Forexample, a FET may be of any suitable type, and may include asemiconducting layer doped with a n-type or p-type material. A source orsource electrode and a drain or drain electrode may be formed in aspaced-apart position on two sides of the semiconducting layer. Thesource electrode and drain electrode may be each doped having anopposite polarity to the semiconducting layer. A suitable dielectriclayer, such as an oxide layer, may underlie the semiconducting layer andthe source and drain. A gate electrode underlies the dielectric layer.In other variations, the gate electrode may be on top of the FET or inits vicinity. A substrate layer made from any suitable material such asplastic or glass serves as a support layer and may underlie the gateelectrode. In certain variations, the semiconducting layer may have asurface that is opposite to the surface to which the dielectric layer isadhered.

Any of the readers described herein may include electrical componentsfor receiving, digitizing and analyzing the analog electrical signalsreceived from the sensors or for controlling the sensor. Such electricalcomponents may include a suitable computer processor or centralprocessing unit, which may be electrically coupled to the electricalpickups of the reader that electrically engage the sensors, where such afeature is provided. The reader may further include suitable storage ormemory, electrically coupled to the processor, for storing computerdata. A suitable display can be included in the reader for displayingdesired information. The display can be a touch screen, for additionallyserving as an input device or terminal. A transmitter or transceiver canbe included in the reader, and electrically coupled therein with aprocessor, for wirelessly transmitting or receiving information betweenthe reader and a suitable remote device.

The reader, alone or in conjunction with another suitable computingdevice, can be calibrated to convert the change in electricalcharacteristic of the electrical component into a concentration level ofthe targeted drug or other substance. In one variation, a suitablealgorithm can be provided in software and stored on a memory of thereader or on a remote device in communication with the reader, orprogrammed onto a chip provided on the reader, so as to permit aprocessor of the reader to manipulate or process the plurality ofmeasurements provided by the sensors on the a device or strip and arriveat an immediate numerical concentration of the targeted substance.

Exemplary Variations of Systems for Detecting a Biological or ChemicalSubstance in Bodily Fluid.

FIG. 1 illustrates a variation of a system 1 for determining a parameterof and/or for detecting a chemical or biological substance in a sampleof bodily fluid. The system includes a substrate 2. One or more activesensors 3 and one or more control sensors 4 are disposed on the surfaceof the substrate 2. For example, FIG. 1 shows three active sensors 3 andthree control sensors 4; however, it is contemplated that any suitablenumber of sensors or sensor pairings may be utilized.

The system 1 also includes an analyzer 10. The analyzer 10 is configuredto analyze the signals received from the first electrical components ofthe active sensor 3 and the second electrical component of the controlsensor 4. The system also includes a reader 20. The reader 20 is coupledto or in communication with the sensors 3, 4 and/or analyzer 10. Thereader 20 is configured to receive an analyzed signal from the analyzer10, and to provide an electronic read-out of the analyzed signal, e.g.,in a visible, user-friendly mode. Optionally, a controller may becoupled to the analyzer or be integrated in the reader to providecontrol and/or programming.

The active sensor 3 includes one or more first electrical componentshaving an electrical characteristic or property. One or morefunctionalized structures (not shown), may be disposed on, near or in avicinity of the substrate 2 or the active sensor 3. The functionalizedstructure and functionalized structure arrangement may include any ofthe functionalized structures described herein, e.g., including thefunctionalized structures illustrated in FIGS. 3A-6 (discussed below).The functionalized structure may interact with (e.g., couple with orbind) the chemical or biological substance. The interaction of thechemical or biological substance with the functionalized structure, orthe interaction of an ion or product released or produced by a reactioninvolving a bound chemical or biological substance, may result in achange in the electrical characteristic or property of the electricalcomponent of the active sensor 3.

The control sensor 4 includes one or more second electrical componentshaving an electrical characteristic. The control sensor 4 is configuredsuch that interaction of the chemical or biological substance with theactive sensor 3 and/or the control sensor 4 does not result in a changein the electrical characteristic of the second electrical components ofthe control sensor 4. For example, the control sensor 4 may bepassivated such that the target chemical or biological substance doesnot bind to or interact with the control sensor 4.

As illustrated with reference to the schematic in FIG. 2, the firstelectrical component of the active sensor 3 produces an active signal30. The active signal 30 is indicative of the changed electricalcharacteristic or the change in the electrical characteristic of thefirst electrical component caused by the interaction of thefunctionalized group or the first electrical component with the chemicalor biological substance in the bodily fluid, or a product (e.g., an ion)released from a reaction involving the chemical or biological substance.For example, the active signal 30 may be indicative of a change incurrent, voltage, capacitance or other electrical characteristic.Simultaneously, the second electrical component of the control sensor 4produces a control signal 40. The analyzer 10 receives, as input, theactive signal 30 from the active sensor 3 and the control signal 40 fromthe control sensor 4. The analyzer produces a comparison (or acomparison signal) between the signals from the active sensor and thecontrol sensor. For example, such a comparison can include adifferential signal 50, being the difference between the active signal30 and the control signal 40. The analyzer may convert the active andcontrol signals from analog to digital. The differential signal 50 isthen transmitted to the reader 20, and used to deduce a parameter, e.g.,concentration of the chemical or biological substance in the sample ofbodily fluid. The reader 20 than provides a read-out based on thedifferential signal, in the form of a value 51 of a parameter of thesubstance, e.g., the concentration of the substance, and/or byindicating whether or not the substance is or is not present 52 in thesample of bodily fluid.

The differential signal 50 may be used to determine a variety ofparameters or characteristics of the chemical or biological substance,or to detect the presence of the chemical or biological substance. Incertain variations, the differential signal 50 may be used to determinethe concentration of various ions present in a target chemical orbiological substance or in the sample of bodily fluid. For example, thedifferential signal 50 may be used to determine the pH of the targetchemical or biological substance.

FIGS. 3A-3B illustrate one variation of a device or substrate 61 havingone or more functionalized structures 65 disposed thereon. The substrate61 includes an active sensor 62 and a control sensor 63. Functionalizedstructures 65 are disposed on the surface of the active sensor 62. Asshown in FIG. 3B, a target chemical or biological substance 67 binds toone or more of the functionalized structure 65 and undergoes a reactionwhich produces one or more ions 68, which diffuse to the surface of theactive sensor 62 where they interact with the active sensor 62 and causea change in an electrical characteristic of the active sensor 62 and/orare detected by the active sensor 62.

FIGS. 4A-4B illustrate another variation of a device or substrate 71having one or more functionalized structures 75 disposed thereon. Thesubstrate 71 includes an active sensor 72 and a control sensor 73.Functionalized structures 75 are disposed on the surface of thesubstrate 71, adjacent to the active sensor 72.

As shown in FIG. 4B, a target chemical or biological substance 77 bindsto one or more of the functionalized structure 75 and undergoes areaction which produces one or more ions 78, which diffuse to or flowover to the surface of the active sensor 72 where they interact with theactive sensor 72 and cause a change in an electrical characteristic ofthe active sensor 72 and/or are detected by the active sensor 72.

The various devices described herein may utilize or work with a varietyof functionalized structures and functionalized structure arrangements,as well as reactions between target chemical or biological substancesand a functionalized structure and/or other reagents, to determineand/or detect various parameters of chemical or biological substances.

FIG. 5 illustrates a variation of an active sensor 82. Functionalizedstructures 85 are disposed on the surface of the active sensor 82. Inthis variation, a first moiety 87 and a second moiety 88 of a targetchemical or biological substance undergo a competing reaction whichproduces ions 89, as the two moieties exchange binding position on thefunctionalized structure 85. The ions 89 then diffuse to the surface ofthe active sensor 82 where they interact with the active sensor 82 andcause a change in an electrical characteristic of the active sensor 82and/or are detected by the active sensor 82.

FIG. 6 illustrates various reactions that may be utilized with thesensor devices and systems described herein, which involve a chemical orbiological substance binding to a functionalized structure disposed on asubstrate, active sensor or in a location remote or separate from thesensor device or substrate. The reactions involve variousfunctionalization schemes as described in more detail below.

In reaction A, a target moiety 91 binds functionalized structure 95,where the binding results in the production of a secondary product 92,which will effect a change in an electrical characteristic of an activesensor.

In reaction B, a target moiety 101 binds functionalized structure 105.The bound target moiety 101 undergoes a reaction with reagent 103, whichresults in the production of a secondary product 102, which will effecta change in an electrical characteristic of an active sensor.

In reaction C, a target moiety 111 binds functionalized structure 115.The bound target moiety 111 binds a secondary functionalized structure114. The binding of the secondary functionalized structure 114 resultsin the production of a secondary product 112, which will effect a changein an electrical characteristic of an active sensor.

In reaction D, a first target moiety 121 binds functionalized structure125. The bound fist target moiety 121 binds a secondary functionalizedstructure 124. The secondary functionalized structure 124 binds a secondtarget moiety 126. The bound second target moiety 126 undergoes areaction with reagent 123, which results in the production of asecondary product 122, which will effect a change in an electricalcharacteristic of an active sensor.

In another example of a reaction, the reaction may include a speciesspecific antibody (e.g. anti mouse, anti rabbit, anti goat, anti guineapig, anti rat, anti lama), which is immobilized onto the sensor surfaceor other location separate from the sensor. Antigen-specific polyclonaland monoclonal primary antibodies raised in, e.g. mouse, rabbit, goat,guinea pig, rat or lama may be added and recognized by the secondaryantibody immobilized to the sensor surface or other surface. For astable interaction, chemical bifunctional cross linkers will be used toirreversibly connect both antibodies.

In other variations, peptides, oligos, ligands or other structures ormolecules may be utilized to provide functionalization to a sensor orother surface. The functionalized structures may be involved or takepart in various reactions, which can be detected or produce productsthat can be detected by the sensor.

In certain variations, the devices, systems and methods described hereinmay provide point-of-care, portable and real-time diagnostic tools. Theymay provide an electronic readout of an enzyme linked immunosorbentassay (ELISA) or other assays to detect various chemical or biologicalsubstances. The electronic components may be configured to transduce orconvert a biochemical binding event or reaction into an electricalsignal, which may be read out. Indirect detection of a freely diffusing,electronically active species produced at the site of a bound chemicalor biological substance may be performed utilizing the describedbiosensor devices. Electronic readout ELISA schemes where an enzymecapable of producing an electronically active species may be utilized.

In one variation, indirect detection may be utilized in a device orsystem described herein where a surface is functionalized with bindingreceptors, such as capture antibodies or engineered proteins, in orderto provide specific binding site. In one example, fins-like tyrosinekinase (sFltl) may be detected. After sFltl is introduced to the device,it binds to the previously immobilized capture Abs. A secondary,biotin-labeled detection Ab is then introduced, which binds to adifferent epitope of sFltl. Streptavidin (SA) conjugated GOx (SA-GOx)tagged enzyme is introduced to bind specifically to the detection Ab.Finally, glucose is introduced and the enzyme-mediated conversion ofglucose to gluconic acid elicits a pH change that can be measured by thesensor.

FIG. 7 shows examples of reactions which cause secondary cascadereactions, which may be utilized with the devices described herein. Thereactions listed in FIG. 7 are merely examples and not meant to belimiting, as other reactions my also cause secondary cascade reactions.

In certain variations, a functionalization area close to the sensorsystem or on the sensor surface where a functionalization and reactiontake place is provided. The functionalization area may include an oxidesurface, nanoparticles, a metal, polymer or any other kind of material.The functionalization can be a protein, antibody or a chemical moietyimmobilized using a linker, which may consist of a chemical surfacemodification, immobilization linkers (such as ProLinker™) or anythingelse which allows to bind the functionalization moiety to the desiredsurface.

In certain variations, the functionalization can be in the form of anassay, e.g. sandwich assay. A reagent may be introduced to the sensorstarting a cascade reaction creating the release of a moiety, e.g. ions.Secondary reagents may freely diffuse to the sensor surface or may bepushed to the surface using a force (e.g. pumps, capillary forces, etc).

In certain variations, a competing reaction may take place exchanging apreviously captured moiety with another one, where the exchange of themoiety releases secondary ions.

In other variations, indirect detection of a freely diffusing,electronically active species produced at the site of a bindingreceptor-immobilized analyte can be performed. The reaction can create achange in the concentration of the released secondary ions. It may causea change in pH (acid or base). Ions that can be released can be but arenot limited to H+, Na+, K+, Cl—, COOH.

Functionalized Sensors Using Engineered Proteins.

Electrostatic- and charge-sensitive devices are often used in liquids asbiochemical sensors. These sensors can directly transduce a biologicalbinding event or biological reaction into an electronic signal in alabel-free manner and are advantageous for sensor technologies demandingdigital readout. Bio-detection in solution is inherently difficult dueto the need of operating in buffered solutions with typically high ionicstrength. Many times the ionic strength of a buffer has a stronginfluence on the sensitivity of the sensor as a result of its inversesquare root relationship with the charge screening distance morecommonly known as the Debye length: the higher the ionic concentrationthe lower is the screening length, which is available to detect thetarget.

Engineered proteins can provide a variety of advantageous features overother receptor molecules currently use for biomolecule detection. Forexample, Affimers™: a) small in size (˜3 nm), b) highly stable(temperature, pH, proteolysis), c) highly selective, d) can beengineered in vitro to most target molecules including small molecules,and d) do not contain endogenous cysteines or functional groups apartfrom engineered ones that can be used for targeted surfaceimmobilization.

When using electrostatic and charge-sensitive biochemical sensors anumber of factors require consideration: i) specificity, ii)selectivity, iii) the distance of the binding receptor to the surface ofthe sensor (or target surface), and iv) uniform surfaceimmobilization—meaning the binding receptor be placed uniformly. Anumber of binding groups and/or functional groups e.g., e.g. antibodies,aptamers, DNA, etc. can sufficiently address specificity andselectivity. However, the distance of the binding receptor the targetsurface and uniform surface immobilization are not well addressed bymany binding groups. Such considerations render many FET based sensorsusing conventional binding receptors molecules with an inability tomeasure detection events in high ionic strength buffers.

For example, the distance of the binding receptor's binding site to thetarget surface is limited by the actual size of the binding receptor.Antibodies for instance are ˜15 nm in size and require bufferedsolutions at high ion concentrations of ˜150 mM for proper performance.However, operating the sensor at these parameters, the charge screeninglength is reduced to a few nm such that the sensor will not sense mostof the binding reactions and signals. Moreover, for most bindingreceptors, including antibodies, an adhesion layer is required forsurface immobilization thereby increasing the distance to the targetsurface even more.

The use of small receptors, such as engineered proteins (e.g., 3 nm insize), can overcome the inability to measure detection events in highionic strength buffers. This permits improved performance sincebiomolecules and their biochemical reactions can occur at ionicstrengths that mimic physiological environments. The use of a bindingreceptor that is closer to the sensor interface dramatically helpsovercome many conventional screening limitations. In addition a morestable binding receptor allows use of the FET sensor in differentsurrounding conditions, such as temperature, lower ionic or variable pHsolutions and thereby facilitates sample preparation and increases thestorage capacity and shelf life of the functionalized sensor.

FIG. 8A illustrates an example of a conventional receptor molecule 130currently use for biomolecule detection. As shown, the screeningdistance or target binding site 132 of a conventional receptor molecule130 can be 15 nm from the immobilization surface of the sensor 150 andthus outside the screening distance. Reducing the distance of thebinding site, for example, using the engineered protein 134 reduces thebinding distance of the target binding site 136 to approximately around3 nm or less. Typically, the screening length is depended on thebackground ion concentration where reducing the ion concentration canincrease the screening length. In any case, the greater the distance ofthe binding site from the surface, the lower the probability ofdetection of a binding event. Therefore, reducing the distance of thebinding site increases the probability that a binding event will even besensed/detected by the sensor and is especially useful where solutionsof high ion concentrations are desired. As noted above, manyconventional receptor molecules also require an adhesion layer toimmobilize the receptor molecule onto the surface of the sensor. Such anadditional layer further increases the binding distance between thebinding site and the surface of the sensor 150. In contrast, the use ofan engineered protein 134 eliminates the need for such an adhesionlayer.

Moreover, use of a binding receptor that is free of endogenousfunctional groups improves uniform surface immobilization—meaning thebinding receptor can be placed uniformly. In many cases, use of abinding receptor that has one or more targeted surface functional groupsengineered directly onto the binding receptor further decreases thedistance to the sensor interface and also allows for a highly uniformsurface.

For example, FIG. 8B illustrates a conventional receptor molecule 130 ashaving one or more endogenous functional groups 138 (e.g., cysteines)throughout the molecule 130. The presence of these endogenous functionalgroups 138 introduces variability in the location of the adhesion siterelative to the surface of the sensor. In certain cases, an additionaladhesion layer is required to improve uniformity. In contrast, theengineered protein 134 depicted in FIG. 8B can include a targetedfunctionalization group 135 to functionalize (or immobilize) theengineered protein 134 onto the desired surface of the sensor in aconsistent or desired manner. Although the targeted functional group 135of the engineered protein 134 is depicted to be located on an end of thestructure 134, one or more functionalization groups can be positioned asdesired on any respective part of the protein structure 134.

In one example electrostatic- and charge-sensitive devices used in aliquid as biochemical sensor can be covered with a thin metal layer,such as gold or other metal that is resistant to corrosion and oxidationin moist air (e.g., a noble metal). In another variation, the samesensor can be covered with nanoparticles, including but not limited tometal nanoparticles, semiconductor layers, an insulator material, or amagnetic material. Further any other shape of a material can be used tocover the sensor. The improved binding receptor (e.g., the engineeredprotein) is then directly attached to the metal layer or nanoparticleusing any conventional process (e.g. thiol chemistry to bind to gold orother metal). In additional variations the sensor does not require anymetal layer. Instead, the sensor can be functionalized on any number ofsurfaces on the sensor or next to the sensor (e.g. oxide surface or anyother type of surface).

FIG. 9 shows one example of a FET sensor 150 used to determine aparameter of a substance in a test sample 140 (typically a liquid). Thecharge sensitive or electrostatic device 152 can be covered with a layersuch as nanoparticles as described above, or a metal, to form anextended gate 154. The extended gate (e.g., the metal or nanoparticlelayer) 154 is then covered with an encapsulation layer 156 that expose aportion of the extended gate 154. The encapsulation layer 156 serves toprotect the device from being exposed to environment factors that woulddamage or degrade the sensor (e.g. such as excess fluids or leakagecurrents). In certain variations, the encapsulation layer 156 covers thewhole sensor chip/strip with an only opening for the extended gate areaor other geometry opening over the sensor area.

The encapsulation layer can be any material that is unaffected or lessaffected by the environment of the test sample or by general conditionsprior to use of the FET sensor 150. The improved binding receptor (suchas the engineered protein) 160 is then directly attached to the extendedgate layer 154 using any known technique (e.g., in cases of a gold gatelayer, thiol chemistry can bind the binding receptor 160 to the extendedgate 154). The sensor can also include a non-conductive material 155,including but not limited to a polymer, dielectric, oxide, nitride, etc.As shown, the exposed gate 154 is in contact with the test sample 140that is functionalized with the engineered protein 160 as describedabove.

FIG. 10A illustrates another example that increases the reliability ofmeasurement of a substance in the test sample 140 using an assessmentand/or comparison of signals between two sensors. For example, the twosensors 150, 151 can both be covered with an encapsulation layer suchthe described metal or nanoparticle layer 154 where only one sensor 150is functionalized with the engineered protein 160 while the other sensor151 is either passivated (with a passivation layer) or is simply notfunctionalized with any binding receptor 160. In the illustratedexample, the second reference sensor 151 can optionally include a secondpassivation layer 162 that makes the sensor 151 just inherent orprevents a reaction with the target moiety. In the illustrated examples,the sensor 150 includes an extended gate 154 having a surface that isfunctionalized with the binding receptor 160, where the functionalizedsurface interacts with the sample 140.

As described above, the functionalized sensor 150 is functionalized withbinding receptors (e.g., the engineered protein) that have at least onefunctional group associated therewith such that the functional group oneach of the binding receptors permits securing each of the bindingreceptors to a first layer of the sensor (in this case an extended gate154) in a desired manner (e.g., the functional groups can secure thebinding receptors in a uniform manner). When placed in contact with thetest sample, the binding receptor interacts with the substance in thetest sample such that interaction of the substance with thefunctionalized sensor results in a change in the electricalcharacteristic of the sensor (in this example the electricalcharacteristic is affected by the change of the extended gate across thesensor channel 158 or in its vicinity). This change causes the sensor152 to produce a changed electrical characteristic that can be detectedupon the application of an electrical stimulus. Such a stimulus caninclude, but is not limited to application of a voltage, current,frequency or other signal allowing detection of the changed electricalcharacteristic from the native electrical characteristic of the sensor.Comparison of the changed electrical characteristic against the nativeelectrical characteristic or the control sensor allows determining theparameter of the substance within the test sample.

FIG. 10B illustrates additional variations of structures for determininga parameter of a substance in a test substance, the device comprisingtwo functionalized sensors 150, 153 and a control sensor 151. Theillustrations are intended to show various configurations of thesensors. As illustrated, the sensors can be placed on substrate layer180 that can comprise any material such as an oxide layer or a polymerlayer. In one example, the substrate 180 comprises silicon or a similarmaterial that allows a voltage to be applied through the substrate layer180 as opposed to the liquid sample 140. Next a dielectric layer 182(such as an oxide material) is applied to permit electrical isolation ofthe various sensors. The sensor contacts 152 are located on thedielectric layer 182 and are bridged with a sensor channel 158. Thesensor channel 158 can comprise any electrically conductive material,structure, layer or coating that allows for electrical communicationbetween the sensor contacts 152. Next, a high-k dielectric material 184(including but not limited aluminum oxide, titanium oxide, zirconiumoxide, yttrium oxide, silicon oxide, tantalum oxide, hafnium oxide andsilicon nitride) is deposited over the gate and/or sensor contacts topermit immobilization of the functionalization layer or bindingreceptors and to reduce potential leakage currents (as shown in sensor150).

As noted above, variations of the device (e.g., sensor 153) can includea high-k dielectric layer 184 with an additional metal and/ornanoparticle layer 186 disposed on the high-k dielectric layer 184 and afunctionalization layer 160 immobilized on the metal/nanoparticle layer186. Alternatively, the layer 182 can be completely removed.

Reference sensor 151 of FIG. 9B illustrates a passivation layer 162located on a high-k dielectric layer 184. However, in additionalvariations, the reference sensor can include a metal/nanoparticle layer186 with or without a passivation layer 162. In either case, thereference sensor will not include a functionalization layer.

As noted above, the test sample can comprise a bodily fluid, or anyother fluid that contains a substance that can be detected upon bindingto the binding receptor.

For example, engineered proteins can be used in conjunction with theactive or functionalized sensor 150 to detect biological and chemicalmolecules from human or other animal bodily fluids, including but notlimited to urine, blood, saliva, tears, ejaculate.

Engineered proteins can be engineered with targeted terminal or internalfunctional groups such as cysteines and immobilized onto a surface(e.g., gold or other noble metal) or suitable nano particles of theactive sensor (or an extension of the active sensor such as an extendedgate) using any known process for immobilization.

In another variation, the engineered proteins can be engineered with N-or C-terminal or internal cysteines and immobilized onto the activesensor surface/nanoparticles modified by self-assembled monolayers(SAMs) using thiol (—SH) chemistry.

Engineered proteins can be immobilized onto the active sensorsurface/nanoparticles coated with carboxylic acid-SAM and using amidecoupling. Alternatively, any other type of SAM layer can be used.

The functionalized sensors can use engineered proteins engineered withterminal streptavidin and immobilized onto (solid supports and) theactive sensor surface/nanoparticles coated with self biotin-SAMs.

Engineered proteins can be engineered with terminal histidine tags andimmobilized on (solid supports and) the active sensor gold surface/goldnanoparticles coated with a Ni2+-NTA (nitrilotriacetic acid) chelatingmoiety. In another application the proteins can be engineered as biotinfusion proteins and immobilized onto streptavidin functionalizedsurfaces (or vice versa). Any number of tags can be engineered dependingupon the desired application.

Immobilization of the engineered proteins can occur via anNH2-functionality onto the SiO2 surface of the active sensor by silanechemistry. In another application, modification of the charge of SiO2can be made by application of short amphiphilic synthetic peptides.

In an additional variation, engineered proteins are generated or used asspecific ligands for bacterial endo- and exotoxins. In anotherapplication, endo- and exotoxin binding (analyte binding) to theengineered protein happens directly on the sensor surfacebiofunctionalized and activated with the engineered capture proteins.

For example FIGS. 11A to 11C provide illustrations of a bindingreceptor, such as the engineered protein, functionalized on the sensor152 (or on the extended gate, not shown).

FIG. 11A shows an analyte 170 (e.g., a substance contained in the testsample) binding to a binding receptor 160 that is immobilized on asurface of an active sensor 153 to produce a change in the electricalcharacteristics electrical component of the sensor.

FIG. 11B illustrates a variation of a functionalized sensor 150 wherethe binding receptor 160 is immobilized on a layer of the sensor 153(e.g., the gate, metal layer, and/or high-k dielectric as describedabove) that allows binding of the analyte 170. However, in thisvariation, a second binding receptor 172 (such as a glucose oxidasetethered to the engineered protein) is introduced to the sensor andbinds to a different analyte 170 binding site. Next, a substance can beadded to enzymatically cause the second binding receptor 172 to generatea reaction that can be measured by the active sensor 153. In the presentexample, the addition of glucose can cause a reaction that generatesgluconic acid and elicits a pH change that can be measured by the activesensor.

FIG. 11C shows another variation or application where endo- and exotoxinbinding to the binding receptor 160 occurs indirectly on a sensorbiofunctionalized with the binding receptor separate from the activesensor 153. As shown, binding receptors 160 immobilized on the sensor153 to bind their target analyte 160. Next, a detection antibody 174 isintroduced that binds to a different site of the analyte molecule 170.Then, a secondary, biotin labeled antibody 176 can be added thatrecognizes and binds to the primary detection antibody 174. Next, astreptavidin (SA)-bound glucose oxidase is introduced to bind biotintethered to the secondary antibody 176. This converts glucose togluconic acid, eliciting a pH change that can be measured by the activesensor in a second location.

In another variation, an actual target molecule (i.e., the substance tobe detected by sensor) can be immobilized on the sensor or substratesurface and the binding receptor (e.g. an engineered protein orengineered scaffold protein, an antibody, peptide etc.) is applied tobind the target molecule. When the actual substance in a test sample isapplied to the sensor, the substance in the test sample competes for thebound engineered protein, thereby releasing the bound receptor andproducing a change in the electric signal of the sensor as noted above.

The binding receptors can be generated or used as specific ligand for anumber of applications, including but not limited to disease causingmicroorganisms including bacteria, yeast, fungi, viruses, parasites; foryeast biomarker, fungal biomarker, viral proteins; a specific ligand fortumor cells or other cells; as specific ligands for disease-related anddrug-related biomarkers (proteins, antibodies, peptides,polysaccharides, lipids, hormones); as specific ligand for smallmolecules (drugs, therapeutics) or drugs subject to abuse; as specificligand for nucleic acids; as specific ligand for heavy metal ions; andas specific ligand for multi-drug resistance proteins causing resistanceto distinct antimicrobials, antibiotics, antifungal drugs, antiviralmedications, antiparasitic drugs, chemicals of a wide variety ofstructure and function targeted at eradicating the organism.

Any of the functionalization schemes and reactions described herein maytake place in a first location remote from or separated from the sensoror device located in a second location. The reaction products or ionsmay then flow to and/or over the sensor or device where detection takesplace.

Each of the individual variations described and illustrated herein hasdiscrete components and features which may be readily separated from orcombined with the features of any of the other variations. Modificationsmay be made to adapt a particular situation, material, composition ofmatter, process, process act(s) or step(s) to the objective(s), spiritor scope of the present invention.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents. Furthermore, where a range of values is provided, everyintervening value between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. Also, any optional feature of theinventive variations described may be set forth and claimedindependently, or in combination with any one or more of the featuresdescribed herein.

All existing subject matter mentioned herein (e.g., publications,patents, patent applications and hardware) is incorporated by referenceherein in its entirety except insofar as the subject matter may conflictwith that of the present invention (in which case what is present hereinshall prevail). The referenced items are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such material by virtue of prior invention.

Reference to a singular item, includes the possibility that there areplural of the same items present. More specifically, as used herein andin the appended claims, the singular forms “a,” “an,” “said” and “the”include plural referents unless the context clearly dictates otherwise.It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

This disclosure is not intended to be limited to the scope of theparticular forms set forth, but is intended to cover alternatives,modifications, and equivalents of the variations described herein.Further, the scope of the disclosure fully encompasses other variationsthat may become obvious to those skilled in the art in view of thisdisclosure. The scope of the present invention is limited only by theappended claims.

What is claimed is:
 1. A method for determining a parameter of asubstance in a sample, the method comprising: providing a substratecomprising an active sensor and a control sensor, wherein the activesensor comprises a first electrical component having a first electricalcharacteristic and at least one functionalized structure in electricalcommunication with the first electrical component, wherein thefunctionalized structure includes a number of binding receptors, whereinthe binding receptors comprise engineered proteins, wherein thefunctionalized structure is coupled to a portion of the substrate in avicinity of the active sensor, and wherein the portion of the substratein the vicinity of the active sensor is an area of the substrateseparated from the active sensor, and wherein the control sensorcomprises a second electrical component having a second electricalcharacteristic; introducing the sample to the substrate, whereinintroducing the sample results in binding of the substance to thebinding receptors and results in the release of ions such that therelease of ions results in a change in the first electricalcharacteristic of the first electrical component; comparing the firstelectrical characteristic of the first electrical component with thesecond electrical characteristic of the second electrical component;using the comparison to determine at least one parameter of thesubstance in the sample; and producing an output of the at least oneparameter.
 2. The method of claim 1, wherein the functionalizedstructure includes a permeable membrane, hydrogel, PVC, or other filter,for allowing only the passage of a target moiety.
 3. The method of claim1, wherein the control sensor includes a passivation structure such as aself-assembled monolayer (SAM), metal layer, polymer layer, or anybiochemically inert material.
 4. The method of claim 1, wherein theparameter of the substance in the sample determined by the comparison ofthe first electrical characteristic and the second electricalcharacteristic is concentration.
 5. The method of claim 1, wherein thesubstance includes a therapeutic , drug, biological moiety, chemicalmoiety, toxin, ion, antibody, peptide, oligonucleotide, pathogen, cells,or natural or engineered ligands.
 6. The method of claim 1, whereincomparing the first electrical characteristic of the first electricalcomponent with the second electrical characteristic of the secondelectrical component includes comparing at least one of a frequency,current, voltage, resistance, impedance, capacitance, conductivity,induction, threshold voltage, transconductance, subthreshold swing,piezo-resistivity, magnetic field, and electrical noise of the firstelectrical component with that of the second electrical component. 7.The method of claim 1, wherein the at least one parameter of thesubstance in the sample is concentration.
 8. The method of claim 1,wherein the first electrical component comprises at least one of atransistor, a capacitor, a resistor, and an inverter.
 9. The method ofclaim 1, wherein the second electrical component comprises at least oneof a transistor, a capacitor, a resistor, and an inverter.
 10. Themethod of claim 1, wherein the active sensor is covered by at least oneof a noble metal layer, a nanoparticle layer, and an organic layer. 11.The method of claim 1, wherein the binding receptor is configured tointeract with the substance to undergo a reaction resulting in therelease of ions and the release of ions affects the first electricalcharacteristic of the first electrical component.
 12. The method ofclaim 11, wherein the reaction resulting in the release of ionscomprises: the binding receptor binding the substance; introducing afirst molecule having a second binding receptor to the active sensor,wherein the second binding receptor binds to a different site of thesubstance bound by the binding receptor; and introducing a secondmolecule to the active sensor, wherein the introduction of the firstmolecule having the second binding receptor and the second molecule tothe active sensor results in an enzymatic reaction that releases ions.